Dusp6 deficiency attenuates neutrophil-mediated cardiac damage in the acute inflammatory phase of myocardial infarction

Dual-specificity phosphatase 6 (DUSP6) serves a specific and conserved function on the dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). We previously identified Dusp6 as a regenerative repressor during zebrafish heart regeneration, therefore we propose to investigate the role of this repressor in mammalian cardiac repair. Utilizing a rat strain harboring Dusp6 nonsense mutation, rat neutrophil-cardiomyocyte co-culture, bone marrow transplanted rats and neutrophil-specific Dusp6 knockout mice, we find that Dusp6 deficiency improves cardiac outcomes by predominantly attenuating neutrophil-mediated myocardial damage in acute inflammatory phase after myocardial infarction. Mechanistically, Dusp6 is transcriptionally activated by p38-C/EBPβ signaling and acts as an effector for maintaining p-p38 activity by down-regulating pERK and p38-targeting phosphatases DUSP1/DUSP16. Our findings provide robust animal models and novel insights for neutrophil-mediated cardiac damage and demonstrate the potential of DUSP6 as a therapeutic target for post-MI cardiac remodeling and other relevant inflammatory diseases.

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For experiments involving qRT-PCR, western blot, immunostaining, ELISA, flow cytometry and RNA-seq analyses, n = 3 was chosen as the minimal replicate number according to the typically minimum standards in the field, shown in previous studies (PMID: 19570514, 23261783, 32130914 and 32076644). Nonetheless, we performed most of these experiments with the sample size no less than 4 for each group. For all animal experiments including MI surgery and echocardiography, as well as the assessments of survival rate and infarct size, we used n = 5 as the minimal replicate number but only for the analyses in Supplementary Figure 12c-d. For other animal experiments in this study, we made the sample size as large as possible, which was determined by the specific situation of animal feeding and availability, to ensure the reliability of the results.
No data exclusion was executed in our study.
We have repeated all presented experiments were at least three times with biological independent samples, and got similar results.
All animals chosen for MI surgery and the subsequent analyses were sex-and age-matched, and were operated and analyzed equally and randomly.
The operator of all echocardiographic analyses was blinded to group allocation during data collection and analysis. For other experiments, blinding was not possible since the primary investigators performed the experiments from the beginning to the end due to the technical nature of the experiments. The specificity of anti-DUSP6 (ab76310) for western blot and flow cytometry was validated using samples from Dusp6-deficient rats and Dusp6 neutrophil-specific KO mice.
The specificity of anti-DUSP6 (TA323084) for immunohistochemistry was validated with samples from Dusp6-deficient rats.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided. anti-Histone H3 antibody (Cell Signaling Technology, 4620): https://www.cellsignal.com/products/primary-antibodies/histone-h3-d2b12-xp-rabbit-mab-chip-formulated/4620 HEK 293T reserved in our laboratory The HEK 293T has been authenticated by STR analysis in our previous study (PMID: 27929112). We did not furtherly authenticate it in this study.
Cell line was not tested for Mycoplasma but no indicaiton of contamination was observed.
No commonly misidentified line was used in this study.
Male Dusp6 mutant and littermate control rats at 8-10 weeks old for MI surgery and other subsequent analyses; Male Mrp8-Cre mice and female Dusp6 floxed mice at 8-10 weeks old to generate Dusp6 neutrophil-specific KO mice; Dusp6 neutrophil-specific KO mice at 8-10 weeks old for MI surgery and other subsequent analysis. Animal room condition was stated in the section of "Method".
No wild animals were used in this study Male mice and rats were used for MI surgery and subsequent analyses in terms ofprevious studies (PMID: 19570514, 23261783, 32130914 and 32076644) . Female animals were used for breeding.
No filed-collected samples were used in this study.
Both rats and mice were raised and handled with the animal protocol (IMM-XiongJW-4) approved by the Peking University Institutional Animal Care and Use Committee, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International